The objective of this proposal is to develop a novel microchip-based CTL assay. MHC-peptide multimers have previously been developed as reagents for CTL detection, enabling the rapid assessment of CTL frequency in HIV infection. Current MHC-peptide-based assays are limited in their application, however, due to cost and technical constraints, as well as by the sensitivity of the assay. Novel developments in the field of microchip technology offer the opportunity to adapt MHC-peptide reagents to a microassay format. Work done under the parent grant has led to the identification of several novel HIV-specific CTL epitopes. Using current assays, however, efforts to identify additional HIV-specific CTL epitopes and to investigate the role of CTL in HIV infection have been hampered by the inability to analyze clinical specimens comprehensively for CTL. We have begun using a comprehensive ELISPOT screen, but application of this assay remains limited by its cost and by the requirement for obtaining large numbers of T cells from each patient. Microchip immunoassays previously developed by Dr. McDevitt's group require nanoliter volumes of blood, and yet have better sensitivity than existing assays. A preliminary collaboration between the Walker and McDevitt laboratories indicates that adapting CTL assays to microchip format will offer similar savings in reagent costs and blood requirements, and may be more sensitive than existing CTL assays. The specific aims of this proposal are: 1) to develop a microbead-based MHC I-peptide multimer for detection of cytotoxic T lymphocytes; 2) to develop a microbead-based assay for detection of cytotoxic T lymphocytes using autologous cells for antigen presentation and 3) to develop chip-based quantitative multiplexed CTL assays. A miniaturized assay for CTL detection in the form of a microchip could potentially represent an enormous advantage in cost savings, technical feasibility and sensitivity over existing assays. Efforts to develop and apply this new technology to HIV immunology would only be possible through the developmental funds available through this grant program.